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Technologies to keep an eye on: alternative hosts for protein
production in structural biology
Francisco J Fernández and M Cristina Vega
The conduct of many trials for the successful production of
large quantities of pure proteins for structural biology and
biotechnology applications, particularly of active, authentically
processed enzymes, large eukaryotic multi-subunit complexes
and membrane proteins, has spurred the development of
recombinant expression systems. Beyond the well-established
Escherichia coli, mammalian cell culture and baculovirusinfected insect cell expression systems, a plethora of
alternative expression systems has been discovered,
engineered, matured and deployed, resulting in crystal, nuclear
magnetic resonance, and electron microscopy structures. In
this review, we visit alternative expression hosts for structural
biology ranging from bacteria and archaea to filamentous and
unicellular yeasts and protozoa, with particular emphasis on
their applicability to the structural determination of high-value,
challenging proteins and complexes.
Address
Center for Biological Research, Spanish National Research Council
(CIB-CSIC), Ramiro de Maeztu 9, 28040 Madrid, Spain
Corresponding author: Vega, M Cristina ([email protected])
Current Opinion in Structural Biology 2013, 23:365–373
This review comes from a themed issue on New constructs and
expression of proteins
Edited by Imre Berger and Lorenz M Mayr
spanning the three domains of life with half of these
entries recording a yeast expression system for their
production (1658 protein chains or 2.20%) (Figure 1a).
These dominant expression platforms share widespread
use and a wealth of available resources (material and
knowledge) and their performance has been demonstrated for many heterologous proteins and multi-subunit
complexes (Figures 2 and 3). Hence, the cost associated
with their adoption for a new protein production project is
generally lower than trying less tested hosts. Nevertheless, research on unconventional expression hosts
represents a valuable addition by shedding light on organisms with orthogonal genomic, metabolic, and recombinant expression properties (Table 1) that can outperform
more established hosts for specific protein families — as
specialty systems. This review aims to summarize stateof-the-art alternative expression hosts including various
bacteria, archaea, filamentous fungi, yeasts, and protozoa,
all of which have been successfully used in structural
biology within the last decade (Table 1 and Figures 1 and
3); inevitably, several hosts will be left out from this
selection: cell-free systems, algae and plants, and bacteria
taxonomically redundant with those already included
(Figure 1b and c). Except when noted otherwise, none
of these expression hosts poses a threat to our health.
Furthermore, we will touch briefly on what the future
may hold for these unconventional hosts.
For a complete overview see the Issue and the Editorial
Available online 5th March 2013
0959-440X/$ – see front matter, # 2013 Elsevier Ltd. All rights
reserved.
http://dx.doi.org/10.1016/j.sbi.2013.02.002
Introduction
Recombinant protein production stands as an enabling
technology in the life sciences and plays a crucial role in
biotechnology and structural biology. At present, Escherichia coli ranks as the most prevalent microbial factory in
structural biology as it has been used to produce more
than 66 724 (88.6%) of all distinct protein chains in the
Protein Data Bank (PDB) with a non-empty descriptor for
‘Expression Host’ (Figure 1a). Insect cells (including
baculovirus-infected insect cells and Drosophila Schneider cells) rank second, accounting for 3499 distinct
protein chains (4.65%), while mammalian cells occupy
the third position with 1911 distinct protein chains
(2.54%) (Figure 1a). The remaining 3102 protein chains
have been expressed in 67 different expression hosts
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Alternative bacterial expression hosts
Pseudomonas
Pseudomonas spp. (typically P. putida, P. aeruginosa, P.
fluorescens and P. alcaligenes) include Gram-negative rods
with strict aerobic metabolism [1] that grow at 30–428C
on inexpensive wastage products like corn molasses and
can be transformed with plasmid DNA by chemical
methods and by plasmid conjugation from E. coli [2].
P. aeruginosa is a moderate potential hazard for staff and
the environment thereby requiring biosafety level 2 (S2)
containment. Its relatively high G + C content (60.5–
66.6% among sequenced species) makes Pseudomonas
an ideal host for the expression of certain restriction
endonucleases [3] (Figure 2a) and naturally G + C rich
gene products, for example, HsaD from M. tuberculosis [4].
Recombinant expression in Pseudomonas may be driven
by temperature (428C) using the temperature-sensitive
PL promoter as well as other promoters such as the IPTGinducible tac promoter, the alkane hydroxylase gene
promoter (alkBp) [5,6], the regulatory control region for
naphthalene and phenanthrene degradation from Comamonas testosteroni (inducible by sodium salicylate) [7], and
Current Opinion in Structural Biology 2013, 23:365–373
366 New constructs and expressions of proteins
Figure 1
Expression hosts
for structural biology
(a)
Yeasts
2.20%
Other (62)
2.01%
Alternative expression hosts
for structural biology
(b)
Mammalian cells
2.54%
Insect cells
4.65%
Non-Methyl.
yeasts
763 chains
639 PDBs
Methylotrophic
yeasts
893 chains
883 PDBs
Bacillus
188 chains
188 PDBs
Other (34)
545 chains
319 PDBs
E. coli
88.60%
Streptomyces
68 chains
68 PDBs
Pseudomonas
63 chains
58 PDBs
% unique protein chains in PDB
(c)
Alternative expression hosts
not covered in the text
Plant/Algae
39 chains
32 PDBs
Cell-free
327 chains
321 PDBs
Mycobacterium
15 chains
14 PDBs
Bacteria (non E. coli) (28.03%)
Yeasts (53.82%)
Other (6)
47 chains
47 PDBs
Aspergillus
96 chains
96 PDBs
Filamentous fungi (4.37%)
Leishmania
2 chains
2 PDBs
D. discoideum
46 chains
41 PDBs
Protozoa (1.52%)
Other (2)
2 chains
2 PDBs
H. salinarum
46 chains
46 PDBs
Archaea (1.52%)
Current Opinion in Structural Biology
Number of expression hosts used for recombinant protein production for structural biology, expressed either as percentage of unique protein chains in
the PDB or as a count of unique chains and PDBs per host. Only PDB entries with a non-empty ‘Expression Host’ field are considered, amounting to
67 690 PDB entries containing 66 722 unique protein chains in total. Absolute counts and frequencies of expression host usage are depicted as pie
charts. When several expression hosts are grouped together as Other, the total number of implied hosts is shown in parentheses. (a) E. coli,
baculovirus-infected insect cells, and mammalian cells represent the dominant expression hosts in the PDB, together accounting for nearly 96% of all
unique chains in protein structures. (b) Summarized statistics for alternative recombinant expression hosts, including yeasts and ‘other’ hosts in (a);
hosts are grouped according to their taxonomic classification and, for yeasts, refined on the basis of their metabolic capacity to assimilate methanol
(methylotrophic and non-methylotrophic yeasts). The area of each pie chart is in proportion to the contribution that each host set makes to the total
number of protein chains expressed with alternative hosts; this frequency is shown underneath the pie charts. (c) As in (b), for alternative hosts not
described in the text (plant/algae and cell-free systems).
the Pm/xylS expression system (Vectron Biosolutions AS)
inducible by toluinic acid and other benzoic acids.
Proteins expressed with this host include, among others,
enzymes, receptors, fimbrial proteins, and heme-containing proteins. So far, 58 PDB entries report Pseudomonas as
expression host of both homologous and heterologous
proteins from other pathogenic bacteria and bacteriophages. Interestingly, non-pathogenic Pseudomonas
species have been used to express and crystallize own
membrane proteins and virulence factors.
Bacillus
B. subtilis and B. megaterium are Gram-positive soil bacteria increasingly used as hosts for intra-cellular and
especially extracellular protein production that have
grown over the past decade into attractive alternatives
to E. coli because their genomes are fully sequenced [8,9],
due to their GRAS (generally regarded as safe) status and
the development of an extensive tool-kit of gene expression plasmids and production strains. Both stable episomal and integrative plasmids are available that can be
Current Opinion in Structural Biology 2013, 23:365–373
efficiently delivered by protoplast transformation or transconjugation. Recombinant expression is typically driven
from the xylose-inducible PxylA [10] or from T7 RNA
polymerase [11] promoters. Even though B. subtilis is
currently more widely used in structural biology, the
strong push to develop B. megaterium as the standard
Gram-positive microbial factory for protein production,
which includes a commercial version (MoBiTec) and
ongoing efforts to characterize it at a systems biology
level [12], may change these numbers in the future. The
198 PDB entries reporting Bacillus as expression host
include diverse glycoside hydrolases, lipases, proteases,
as well as insecticidal peptides, virulence factors, for
example, anthrax lethal factor and Clostridium difficile
toxin A [13] (Figure 2b) and phage DNA polymerases.
Streptomyces
Several Streptomyces strains have been used for the production of proteins for 67 PDB entries, mostly S. lividans
TK24 because of its exceptionally low level of extracellular proteases. These mycelial Actinobacteria are versatile
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Expression
host a
Bacteria
(simple,
robust, lowest
cost)
Escherichia coli
b
Gene regulatory sequences
(activation) c
Expression
level e
Vectors f
Proteolytic Phosphor Glycosylation i PTMs j
processing g ylation h
Learning
curve k
Time
(wk) l
Economic
cost m
Recommended use n
High (inclusion Episomal
bodies)
No
Rare
No
No
Reference
1–2
Other
Pseudomonas
Enterobacteria spp.
1.0–2.0
High
Episomal
No
Possible
No
No
<3 months
2–4
Firmicutes
60–67% PL (switch to 428C)
Ptac (IPTG) PalkBp (sodium
salicylate)
Pm/xylS (toluinic/benzoic ac.)
Bacillus spp.
39–43% PxylA (xylose)
PT7 (IPTG)
Streptomyces
72% Ph. starvation promoter
spp.
Erythromycin promoter
Nitrilase promoter
Hybrid T7 promoter
Mycobacterium
67% Acetamidase (acetamide)
spp.
0.6–1.0
High
(secretion)
High
(secretion)
Episomal
No
Possible
No
No
<2 month
1–3
Episomal
No
Possible
No
No
3–6 months
2–4
2.0–3.0
Low-high
Episomal
No
Possible
No
No
<3 months
2–4
Halobacterium
halobium
65% Strong constitutive promoter
4.0–8.0
High
(membrane)
Episomal
Some
Possible
No
No
3–6 months
2–4
Methylotrophic
(P. pastoris, H.
polymorpha)
41% PAOX1 (methanol)
PFLD (acetamide)
PGAP (constitutive)
1.5–3.0
Low-high
(secretion)
Integrative Some
Some
High
Some <6 months
3–6
Non36–39% PGAL1/PGAL10 (galactose)
methylotrophic
Acetamidase (acetamide)
(S. cerevisiae,
Glycolytic (constitutive)
K. lactis)
Aspergillus spp. 49–51% IPTG inducible
Hypocrea/
Glycolytic (constitutive)
PglaA (xylose) PalcA (ethanol)
Trichoderma
Human estrogen (estrogen)
spp.
58% 18S rRNA locus (constitutive)
Leishmania
terantolae
PT7/tetO (tetracycline)
Dictyostelium
23% PActin-15 (constitutive)
discoideum
PDiscoidin I (constitutive)
PrnrB (UV inducible)
Sf9, Sf21, Hi-5 35–41% p10, polh (viral life cycle)
1.3–2.0
Low-high
Episomal/ Some
integrative
Some
High
Some <3 months
2–4
3.0–4.0
Low-high
(secretion)
Episomal/ Some
integrative
Some
High
Some <6 months
2–6
6.0–13
Low-high
Integrative Some
Extensive Complex
High
<6 months
2–4
Episomal
Extensive Core
High
<6 months
2–4
Yes
Complex
Yes
6–12 months 3–5
Moderate-high General purpose
CHO, HEK293
14–36
Yes
Complex
Yes
<12 months 3–8
High
Mycobacteria
Archaea (low cost, Euryarcheota
good for some
membrane
proteins)
Unicellular
Eukarya
fungi (yeast)
(glycosylation,
PTMs)
Filamentous
fungi
Protozoa
Current Opinion in Structural Biology 2013, 23:365–373
BVS
Mammalian
cells
50% PT7, Ptac (IPTG)
PBAD (arabinose)
tD (h) d
0.3–0.5
Actinobacteria
a
%GC
41% SV40, CMV (constitutive)
4.0–6.0
4.0–12.0 Low-high
16–72
Some
Low-high
Bacmid
Yes
(cytosol)
Low-moderate Episomal/ Yes
(secretion)
integrative
Reference
Small proteins Single
($1000/protein) domains
Antigens
Low-moderate Specific receptors
Fimbrial proteins
Heme proteins
Low
Secreted enzymes
Virulence factors
Low-moderate Antibiotic peptides
Antibiotic biosynthesis
and drug-modifying
enzymes
Low-moderate Mycobacterial
antigens and
cell-wall enzymes
Low
Bacteriorhodopsin
GPCRs
Low-moderate Secreted proteins
Complement factors
Membrane proteins
Peroxysome proteins
Low
Kinases
Nuclear proteins
Membrane proteins
Antibodies
Low-moderate Secreted enzymes
Glycoenzymes
Moderate-high General purpose
Abundant PTMs
Moderate-high Glycoproteins
Motor proteins
General purpose
Organisms exploited for recombinant expression of homologous or heterologous proteins (details of each host are expanded on the text), organized according to their taxonomy and, for yeasts, to their capacity to thrive in methanol.
%GC: GC content (percentage) of complete genome. GC content may affect protein expression indirectly via codon bias and availability of tRNA pools.
c
Selection of the most frequently used promoters and their activation (inducible or constitutive).
d
tD: Doubling time (h). Faster dividing expression hosts will generate more biomass per unit time; exact doubling times depend on temperature, media composition, and genetic background.
e
Qualitative scale aimed at describing the potential for an expression host to achieve high-level expression levels; it is highly dependent on the type of protein being expressed. The rough scale translates into an expected yield in mg per liter culture:
low, 0.1–5.0 mg/L; moderate: 5.0–50.0 mg/L; high: >50 mg/L. Further information is shown between parentheses.
f
Typical vectors used to drive recombinant expression, either episomal (stably maintained extrachromosomally) or targeted for genomic integration (integrative).
g
Capacity to perform mammalian-style proteolytic processing of the expressed recombinant protein.
h
Capacity to perform mammalian-style phosphorylation of the expressed recombinant protein.
i
Capacity to glycosylate recombinant proteins; high: abundant glycosylation of the high mannose type; core: mammalian-style core glycosylation; complex: full or nearly full mammalian-type glycosylation.
j
Overall capacity for complex PTMs in comparison to mammalian cells.
k
Learning curve tries to capture the approximate time it would take a scientist with previous exposure only to E. coli expression to become proficient (i.e. productive mostly independently) in the other expression system; no expert guidance is
assumed since intensive next-door expert training or translational access programs to dedicated expert facilities would certainly accelerate learning these technologies.
l
Time: time in weeks for an experienced user from start of project to the first large-scale expression culture, hence spanning cloning, transformation and selection, small-scale expression trials, and large-scale expression culture.
m
Economic cost is a complex variable that depends on any particular target protein, dedicated person-months and length of the overall process (from clone to purified protein), gene synthesis (or not), target yield, fermentation scale, and the
combined costs of media, antibiotics, inducers, and other reagents. Hence, the values shown are merely a rough guide that might oscillate (upwards and downwards). Low, comparable to the cost involved in the soluble expression of a single-domain
protein in E. coli (estimated at $1000 from cloning to expressed protein); moderate, 2–5-fold higher overall cost; high, >5-fold higher cost.
n
Recommended use: highlighted/recommended applications suggesting potential areas with the greatest promise for further development for each host.
b
Technologies to keep an eye on: alternative hosts for protein production in structural biology Fernández and Vega 367
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Table 1
368 New constructs and expressions of proteins
Figure 2
(a)
(b)
(c)
C. difficile Toxin A glucosyltransferase
bound to UDP-glucose
expressed in B. megaterium
(PDB 3SRZ)
Pacl-DNA (5′-TTAATTAA-3′)
in the high %GC P. alcaligenes
(PDB 3M7K)
(d)
(e)
Mt Oxoacyl-[acyl-carrier protein] synthase 1
expressed in M. smegmatis
(PDB 2WGD)
(g)
(f)
T90A/D115A Bacteriorhodopsin mutant
expressed in H. salinarum
(PDB 3COD)
(h)
At H+-ATPase proton pump
expressed in S. cerevisiae
(PDB 3B8C)
O-carbamoyltransferase TobZ
expressed in S. lividans
(PDB 3VEN)
Human complement receptor 2 (SCR1-2)
expressed in P. pastoris
(PDB 1LY2)
(i)
Glucohydrolase GH61 isoenzyme A
expressed in A. oryzae
(PDB 2YET)
Human Superoxide dismutase [Cu-Zn]
expressed in L. tarentolae
(PDB 3KH4)
(j)
Myosin
Myosin
Myosin
Rigor actin-tropomyosin-myosin complex
expressed in rabbit (actin), D. discoideum (myosin), and E. coli (tropomyosin)
(PDB 4A7F)
Current Opinion in Structural Biology
Gallery of crystal structures where proteins were overexpressed using an unconventional platform. (a) Restriction endonuclease PacI complexed with
its DNA recognition sequence expressed homologously in Pseudomonas alcaligenes (3MK7) [3]. (b) Glucosyltransferase domain from Clostridium
difficile toxin A bound to UDP-glucose expressed in Bacillus megaterium (3SRZ) [13]. (c) O-carbamoyltransferase TobZ from Streptoalloteichus
tenebrarius expressed in Streptomyces lividans (3VEN) [14]. (d) KasA protein (3-oxoacyl-[acyl-carrier-protein] synthase) from Mycobacterium
tuberculosis expressed in Mycobacterium smegmatis (2WGD) [17]. (e) T90A/D115A mutant bacteriorhodopsin expressed homologously in
Halobacterium salinarum (3COD) [22]. (f) D73 C-terminally truncated plasma membrane proton pump from Arabidopsis thaliana expressed in
Current Opinion in Structural Biology 2013, 23:365–373
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Technologies to keep an eye on: alternative hosts for protein production in structural biology Fernández and Vega 369
Figure 3
(a)
Factors to consider when selecting
an expression host for structural biology
(c)
Recommended use of alternative expression hosts
Gene of interest
gene features
protein features
localization
• source
• size
• %GC
...
• multi-domain
• enzyme
• PTMs
• glycosylated
...
• membrane
• nuclear
• peroxysome
• secreted
...
(c)
Generalists vs. Specialists
(number and % heterologous proteins expressed)
Bacteria
Pseudomonas
Bacillus
Streptomyces
Mycobacterium
Archaea
H. salinarum
Eukarya
Methylotrophic yeasts
Non-methyl. yeasts
Filamentous fungi
Protozoa
Leishmania
Dictyostelium
Methylotrophic yeasts
(883 PDBs)
• secreted enzymes
• cytosolic enzymes
• glycosylated proteins
• complement factors
Non-methylotrophic
yeasts (639 PDBs)
• fast screening
• nuclear proteins
• membrane proteins
• functional assays
(i) bioinformatics analysis
(ii) experimental knowledge
Unique chains
PDBs
11/63 (17.5%)
34/188 (18.1%)
25/68 (37.3%)
0/15 (0.0%)
10/58 (17.2%)
34/188 (18.1%)
25/67 (37.3%)
0/14 (0.0%)
0/46 (0.0%)
0/46 (0.0%)
887/892 (99.4%)
561/763 (73.5%)
78/137 (59.4%)
875/883 (99.1%)
541/639 (84.6%)
78/137 (59.4%)
2/2 (100.0%)
1/46 (2.2%)
2/2 (100.0%)
1/46 (2.2%)
Bacillus
(188 PDBs)
• secretion
• virulence factor
L. terantolae
(2 PDBs)
Mycobacterium
(6 PDBs)
Aspergillus
(96 PDBs)
• secretion
• lipases/proteases
• glycoenzymes
Streptomyces
(67 PDBs)
• antibiotic peptides
• antibiotic synthetases
• drug-modifying enzymes
H. salinarum
Pseudomonas
(46 PDBs)
(58 PDBs)
• bacteriorhodopsin
• fimbrial proteins
(including mutants)
• Ps. receptors
• GPCRs
• heme proteins
• high %GC endonucleases
D. discoideum
(41 PDBs)
• motor proteins
• glycoproteins
Current Opinion in Structural Biology
Selection of a suitable alternative expression host for a protein expression project. (a) Features of the target gene and the encoded protein that bear on
the choice of expression host can be collected from bioinformatics analysis and known experimental facts (own research or published). Some of those
features may play crucial roles in tipping the balance towards an unconventional host, for example, if the target protein is secreted to the extracellular
medium. (b) Number and percentage of heterologous proteins expressed with several alternative hosts, expressed as unique chains or as number of
PDBs. A host used to express 50% or more of heterologous proteins is said to be a generalist, otherwise it is considered to be a specialist. (c) Pie chart
showing the same alternative hosts as in (b), with section areas representing frequency of use in the PDB. To aid in choosing from them, guidelines are
shown underneath each expression host regarding the recommended use for that host.
hosts for the production of secondary metabolites
accounting for half of all commercialized antibiotics
and are especially indicated for antibiotic peptides and
enzymes involved in antibiotic biosynthesis and drug
modification, for example, the O-carbamoyl transferase
TobZ [14] (Figure 2c). Several strong promoters are
available controlled by phosphate starvation, inducible
by erythromycin, and the hyper-inducible nitrilase promoters [15], as well as hybrid promoters employed to
drive expression from a T7 promoter to very high levels
[16]. Hence, Streptomyces strains appear particularly well
suited for the recombinant production of antibiotic and
drug-modifying enzymes in addition to peptides and
small-molecule antibiotics and drugs.
Mycobacterium
Two fully sequenced, non-pathogenic species for human,
M. smegmatis and M. vaccae, have been successfully
exploited for the production of M. tuberculosis proteins in
work spearheaded by the M. tuberculosis structural genomics consortium that has resulted in 5 and 1 deposited
PDB entries, respectively. Even if those entries represent
only a small fraction of the >1000 structures concerning M.
tuberculosis proteins, they include Mycobacterial antigens,
Saccharomyces cerevisiae (3B8C) [33]. (g) Unliganded human CD21 (complement receptor 2, CR2) SCR domains 1-2 expressed in Pichia pastoris
(1LY2) [39]. (h) Glucohydrolase 61 (GH61) isozyme A (post-translationally modified by methylation and two disulfide bridges) from Thermoascus
aurantiacus expressed in Aspergillus oryzae (2YET) [50]. (i) Human Cu/Zn superoxide dismutase (SOD1) expressed in Leishmania tarentolae (3KH4)
[53]. (j) Actin–tropomyosin–myosin rigor complexes expressed in Dictyostelium discoideum, rabbit, and E. coli (4A7F) [60].
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Current Opinion in Structural Biology 2013, 23:365–373
370 New constructs and expressions of proteins
iron-superoxide dismutase, and cell-wall and specific lipid
biosynthetic enzymes [17] (Figure 2d) that may have
required elaborate strategies for their expression in E. coli.
Vectors for recombinant expression in M. smegmatis usually
drive expression from the strong acetamidase promoter,
which is induced by acetamide at mid-log phase and peaks
at 24–36 hours [17,18]. M. smegmatis and M. vaccae
represent invaluable additions to the protein expression
toolbox of researchers dealing with enzymes involved in M.
tuberculosis specific pathways.
Archaeal expression hosts
Bacteriorhodopsins are model systems for light-harvesting membrane proteins and can be obtained in large
amounts from the natural source owing to their strong
constitutive expression. However, investigation of
mutant bacteriorhodopsins cannot simply rely on the
natural source or time-consuming and labor-intensive
genome mutagenesis, therefore providing an incentive
to developing recombinant systems to express bacteriorhodopsins. One such system uses the extreme halophilic
Euryarcheaota Halobacterium salinarum (syn. halobium)
[19–21], which has been harnessed in 46 PDB entries,
including several mutant structures [22] (Figure 2e);
another extreme halophilic archaeon, Natromonas pharaonis, has been used at least once (PDB 1JGJ). Shuttle E.
coli–Halobacterium vectors have been constructed that
contain the pHH9 H. salinarum replication origin, E. coli
tetracycline resistance gene (Tetr) and the H. volcanii
novobiocin resistance gene (Novr) [23]. In addition,
human G-protein coupled beta2-adrenergic receptor
has also been successfully expressed in H. salinarum
[24], thus providing proof-of-concept of the potential
usefulness of these archaeal hosts for the production of
mammalian membrane proteins in general and GPCRs in
particular.
metabolic flux analysis in yeast render them promising
tools for present and future biotechnology.
Non-methylotrophic yeasts
763 distinct protein chains in 639 PDB entries list a nonmethylotrophic yeast as the recombinant host for expression. Of those, Saccharomyces cerevisiae is the most frequently used (741) followed by Yarrowia (11),
Kluyveromyces lactis (8) and Schizosaccharomyces pombe
(3). Nearly 50% of them are human soluble proteins
and protein complexes [32], but there are also membrane
proteins [33] (Figure 2f) and even viral capsids completely reconstituted in yeast.
Methylotrophic yeasts
These yeasts can feed on methanol as sole carbon and
energy source and represent the yeast-based expression
host with the highest adoption rate; especially because of
the high cell densities they reach in mineral minimum
medium and the strength of the alcohol oxidase 1 (AOX1)
and methanol and nitrogen-regulated FLD [34] promoters or the constitutive GAP promoter [35]. Heterologous
production of fungal, plant, mammalian and human
proteins is well-documented, especially by secretion to
the extracellular medium. Highlight examples of this
strategy are the full-length versions of innate immunity
proteins and protein complexes, for example, complement control proteins and complement receptors, including the human decay-accelerating factor (DAF,
CD55) [36], and human complement receptors 1 (CR1)
[37,38] and 2 (CR2, CD21) [39] (Figure 2g). To date, P.
pastoris has been employed in about 880 PDB entries
since first reported in the 1994 NMR structure of tick
anticoagulant peptide [40].
Filamentous fungi
Aspergillus
Yeasts expression systems
These single-celled fungi have been extensively
researched as hosts for heterologous protein production,
including non-methylotrophic genera as Saccharomyces,
Schizosaccharomyces, Kluyveromyces [25], and Yarrowia
[26], and methylotrophic genera as Pichia pastoris (syn.
Komagataella phaffii) [27,28] and Ogataea (Hansenula) polymorpha (syn. P. angusta) [29,30,31]. Attractive features
include well-known genetics and metabolism, easy to
perform gene knockout and gene replacement, availability of strong promoters (constitutive as well as tightly
controlled and inducible), and fast growth to very high
cell densities on inexpensive media (in microtiter plate,
shake flask and bioreactors). Being lower eukaryotes,
yeasts can perform many post-translational modifications
as higher eukaryotes and in particular possess a secretory
pathway capable of correct protein processing and highmannose glycosylation. With serious efforts well underway to engineer humanized glycosylation in several yeasts
platforms, available tools for whole genome analysis and
Current Opinion in Structural Biology 2013, 23:365–373
In contrast to yeasts and the other microbial factories
described here, Aspergillus is a multicellular organism, a
filamentous fungus. Several Aspergillus species, chiefly A.
niger and A. oryzae, are workhorses in many biotechnology
applications [41,42] and have also been used in structural biology. Their amenable genetics and strain optimization features, an unmatched capacity for secreting
homologous and heterologous enzymes, as well as
detailed knowledge of its industrial-scale fermentation,
make Aspergillus an attractive expression host. Random
mutagenesis screenings aimed at abolishing the proteolytic secretoma have also succeeded in isolating mutants
with 98% less proteolytic activity than the wild type,
apparently by the gene disruption of a fungal-specific
master regulator of protease gene expression [43].
Although harder to transform than yeasts, methods for
the efficient transformation of Aspergillus have been
developed that include Agrobacterium tumefaciensmediated DNA transfer [44] and transformation of protoplasts after enzymatic cell wall degradation [45]. Both
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Technologies to keep an eye on: alternative hosts for protein production in structural biology Fernández and Vega 371
constitutive and inducible promoters are available for A.
niger expression of foreign proteins, with inducible promoters being preferred because of the beneficial effect on
protein yield of decoupling biomass production and
protein expression (e.g. in the production of toxic
proteins). Two inducible promoters are commonly used,
the A. niger glucoamylase gene promoter PglaA repressed
by xylose and highly induced when maltose or starch is
used as single carbon source [46] and the A. nidulans
alcohol dehydrogenase gene promoter PalcA, induced by
ethanol and derivatives [47]. Recently, promising human
estrogen inducible promoters ensuring both tight regulation and a linear inducer response have been developed
[48]. Glycosylation in Aspergillus is of the high mannose
type and both N-linked glycosylation and O-linked glycosylation are efficiently catalyzed. The Aspergillus toolbox was until recently lacking in expression vectors
encoding fusion tags for purification [49]. To date, 130
NMR and X-ray crystal structures of fungal proteins
expressed in A. niger and A. oryzae have been reported,
for example, [50] (Figure 2h), demonstrating the feasibility of using filamentous fungi for protein production for
structural biology applications, which require very homogeneous, pure and abundant protein. In fact, some of the
above issues have been addressed and solved, the greatest
success having been reached using synthetic gene
sequences which were codon optimized for A. niger [51].
Protozoa
Leishmania tarentolae
Several characteristics of L. tarentolae make it a convenient microorganism for the production of authentically
processed eukaryotic proteins: this trypanosomatid protozoan infects lizards and is non-human pathogenic;
therefore it is innocuous and only requires biosafety level
1 laboratories; and it is a robust organism that grows
optimally at 268C and can be kept under sterile conditions
outside the hood using a flame. Constitutive or inducible
vectors for intracellular or secretion expression are commercially available with optimized splicing signals for
expression of heterologous proteins [52], which can be
either stably integrated into the genome (typically into
the strongly transcribed 18S rRNA locus) or maintained
episomally. L. tarentolae cells, transformed by electroporation, can grow in adherent culture or be cultivated to
higher densities in shaking cultures (5 108 cells/ml) in
inexpensive, bacteriological media without serum.
Examples of expressed proteins include human erythropoietin and interferon-gamma. To date, two PDB entries
report having used L. tarantolae as expression host in two
different crystal forms of human Cu/Zn superoxide dismutase [53] (Figure 2i).
Dictyostelium discoideum
The social amoeba D. discoideum is recommended as a
non-mammalian model organism for functional analysis of
sequenced genes by the National Institutes of Health
www.sciencedirect.com
(Bethesda, MD, USA) since it provides an intermediate
level of complexity between yeasts and plants and
animals [54]. More than 30 genes orthologous to human
disease genes have been found in D. discoideum 34 Mb
genome, which shows a higher degree of gene conservation with higher eukaryotes than with fungi [55]. With an
A + T content of >75%, the expression of certain genes
has been enhanced by gene optimization (adjusting for a
similar codon preference) and using authentic signal
sequences [56,57]. Foreign DNA is efficiently delivered
to D. discoideum by electroporation and transformants can
be cultured xenically on a bacterial food source or axenically on simple salt medium, with a doubling time of 4–
12 hours that exceeds the growth rate of mammalian cells.
D. discoideum performs mammalian-style N-linked and Olinked glycosylation but cannot accomplish complex
terminal glycosylation since it lacks the ability to add
galactose, N-acetyl galactosamine, or sialic acids [58,59].
In fact, several therapeutic glycoproteins have been successfully expressed in D. discoideum including rotavirus
VP7, muscarinic receptor m2 and m3, antithrombin III,
gonadotropins, or erythropoietin. The most remarkable
applications of D. discoideum in structural biology concern
the production of wild-type, chimeric, and mutant motor
proteins, for example, the actin–tropomyosin–myosin
rigor complexes [60] (Figure 2j) and dynein [61].
Conclusion
Success in recombinant protein expression for structural
biology involves making crucial choices about construct
design, coexpression partners, culture parameters, and
induction regime, among many others — including
switching to unconventional expression hosts that may
be particularly well-suited to the target gene for various
reasons (Figure 3). These lesser known organisms, which
can accomplish expression of heterologous proteins (as
general purpose hosts or ‘generalists’) as well as revealing
themselves most valuable when over-producing their own
proteins or specific classes of macromolecules (as ‘specialist’ hosts) (Figure 3b), include Gram-negative and Grampositive bacteria, extreme halophilic archaea, and eukaryotic fungi (yeast and filamentous fungi) and protozoa. As
demonstrated in more than 3100 distinct PDB protein
chains, these hosts can efficiently generate high yields of
proteins endowed with correct fold, activity, and posttranslational modifications, and possess sufficient quality
for structural and biophysical analysis. Growing access to
those hosts via commercial or academic ready-to-use
toolkits should facilitate their comparative screening
and the solid experimental assessment of their relative
merits for the production of different classes of proteins
and protein complexes.
Acknowledgements
This work was supported by the EC project ComplexINC (Framework
Programme 7 (FP7) under grant agreement no. 279039) and Comunidad de
Madrid project Complemento-CM (S2010/BMD-2316).
Current Opinion in Structural Biology 2013, 23:365–373
372 New constructs and expressions of proteins
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